1. Field of the Invention
This invention relates to radioimmunoassay methods for the quantitative determination of the iodothyronine hormones triiodothyronine (T-3) and thyroxine (T-4) in biological fluids such as serum. The present invention provides a unique method for the combined or concurrent radioimmunoassay of these hormones in serum or plasma wherein the same radioisotope is comprised in the radiolabeled T-3 and T-4 reagents used in the assay.
The secretion of T-3 and T-4 by the thyroid gland is controlled by thyrotropin (TSH), a thyroid-stimulating hormone from the anterior pituitary which in turn is controlled by a thyrotropin releasing hormone (TRH) secreted by the hypothalamus [Evered, Clinics in Endocrinol. Metab. 3, No. 3, W. B. Saunders and Co., Ltd. (1974)]. The first true assay for thyroid hormone was a competitive protein binding assay (CPBA) used by Ekins, Clin. Chim. Acta 5:453(1960), in which he used a radioactive tracer and thyroxine-binding globulin (TBG) as the primary hormone binder. The CPBA technique grew in popularity through the work of Murphy and Pattee [J. Clin. Endocrinol. Metab. 24:187-196(1964)] and others, and was the predominant testing method for T-4 until the advent of the radioimmunoassay (RIA).
Previously, T-4 was thought to be the primary hormone regulating metabolism. Little was known about the clinical significance of T-3 in the presence of the relatively large physiological levels of T-4. Gross and Pitt-Rivers [Lancet 1:439-441(1952)] were the first to identify T-3 as a thyroid hormone. With the advent of radioimmunoassay techniques [Brown et al, Nature 226:35(1970) and Neuman et al, J. Clin. Invest. 46:1346-1355(1967)], the measurement of serum T-3 has gained wide recognition as an important thyroid function test [Larson, Metabolism 21:1073-1092(1973); Hoffenburg, Clin. Endocrinol. 2:75-87(1973) and Harvard, Brit. Med. J. 1:553-556(1974)]. As much as 40% of circulating T-3 may be derived from the deiodination of circulating T-4 [Braverman et al, J. Clin. Invest. 49:855-864(1970)], and it has been estimated that as much as two-thirds of the normal thyroid hormonal effect can be attributed to T-3 [Sterling, New Eng. J. Med. 284:271-272(1971)].
The levels of T-3 and T-4 are normally maintained by their negative feedback effort in controlling the output of TSH. Generally, changes in T-3 levels are paralleled by similar changes in T-4, but in some circumstances the changes are independent. For example, a form of hyperthyroidism, T-3 toxicosis, has been described by Hollander [Trans. Assoc. Am. Physicians 81:76-91(1968)] and Sterling et al [J. Am. Med. Assoc. 213:571-575(1970)] in which the T-3 level is elevated while the T-4 level remains normal. Clearly, a single radioimmunoassay that would quantitate both total T-3 and T-4 in serum or plasma would be a convenient and valuable contribution to the comprehensive assessment of a patient's thyroid function status.
2. Brief Description of the Prior Art
Formerly, radioimmunoassay techniques were used primarily for the measurement of a single substance. More recently, since methods were developed to produce antisera having greater specificity, interest has grown in the application of RIA technology to concurrent measurement of more than one antigen. The present invention provides an RIA method for the combined measurement of T-3 and T-4 using the same radioisotope as tracer. A combined RIA method for these hormones must demonstrate considerable specificity since these two materials differ in structure by only a single iodine atom.
U.S. Pat. No. 3,659,104 discloses a method of measuring serum thyroxine employing an alkaline crosslinked dextran gel column to dissociate and separate the T-4 from serum protein. The method is unique in combining the extraction and saturation analysis steps on the same test device. This technique is applied to the determination of serum T-3 in U.S. Pat. No. 3,961,894. Neither of these patents suggests the combined determination of T-3 and T-4 nor any method capable of accomplishing such an assay.
A radioimmunoassay method for T-3 or T-4, or for T-3 and T-4, present in unextracted serum is described in U.S. Pat. No. 3,928,553. In the method, barbital or salicylate is used to inhibit thyroid hormone binding by serum prealbumin, and dilantin, salicylate, merthiolate, tetrachlorothyronine or 8-anilino-1-naphthalene sulfonic acid is used to inhibit the binding by thyroxine binding globulin. The method uses isotopically-labeled triiodothyronine (.sup.125 I T-3) and thyroxine labeled with a different radioisotope (.sup.131 I T-4). Separation of antibody-bound and unbound T-3 and T-4 is carried out by adsorption of the unbound portion on dextran coated charcoal, followed by centrifugation. No mention is made of the specificity of the assay for T-3 and for T-4 and therefore their independence in the assay is undescribed.
A very similar procedure for determining both T-3 and T-4 using T-3 and T-4 labeled with different radioisotopes (.sup.125 I and .sup.131 I, respectively) is described by Ljunggren et al, Acta. Endocrinol. 81:487-494(1976). Detectable levels of contaminant T-3 were found in the standard T-4 solution requiring a correction factor. Also, the influence of .sup.131 I on the counting of .sup.125 I was found to be in the area of 20 percent.
Habermann et al, J. Clin. Chem. Clin. Biochem. 14:595-601(1976), describe a combined RIA procedure for T-3 and T-4 in urine with no application to assaying serum or plasma. In urine, T-3 and T-4 are present in similar concentrations whereas T-3 appears in much lower concentrations than T-4 in serum. Also, protein binding of T-3 and T-4 is a significant factor to deal with in assays of serum but not so for urine assays. In the Habermann et al method, a sample of a 24-hour urine pool is applied to a dextran gel column equilibrated with a highly alkaline solution. Radiolabeled (.sup.125 I) T-3 and T-4 are added to the column which is then washed with buffer at pH 7.4. After incubating T-3 antibody on the column for 2 hours, the antibody bound T-3 is eluted with the buffer. Then T-4 antibody is incubated on the column for 2 hours and antibody bound T-4 eluted with buffer. The radioactive T-3 and T-4 eluates are then measured separately and compared to standard results. This method could not take advantage of measuring radioactivity on the columns because of the presence of radiometrically indistinguishable .sup.125 I T-3 and .sup.125 I T-4 on the column throughout the method.
U.S. Pat. No. 3,952,091 describes a simultaneous multiple radioimmunoassay providing a qualitative indication of the presence of one or more of a specific group of antigens. The assay is a simple screening test which cannot distinguish one antigen from another and cannot provide quantitative results.